With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 60% of all human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. The Hertel laboratory (Principal Investigator) has successfully used quantitative approaches to uncover important regulatory aspect of the pre-mRNA splicing reaction. The Baldi laboratory (Collaborator) has been instrumental in the development of bioinformatics approaches required for the analysis of large datasets. In this proposal, the expertise of both groups will be combined to determine the mechanisms of alternative splice-site activation, one of the most frequent forms of alternative pre-mRNA splicing. Biochemical assays will be complemented by computational analyses of large EST databases to provide a molecular understanding for alternative splice-site activation. The long- term goal of this research proposal is to understand the mechanisms by which the splicing machinery correctly identifies exons and faithfully removes intervening sequences. The experiments proposed in this application have two major goals: To characterize the activation of alternative 5' splice sites (Specific Aim 1) and to characterize the activation of alternative 3' splice sites (Specific Aim 2). Preliminary computational analysis of the human genome demonstrated an unusually high frequency of alternative 5' splice-site activation 4 nucleotides upstream or downstream of the dominant 5' splice site. However, it is not clear whether these overlapping splice sites are activated stochastically or whether they are induced by regulatory elements. In Specific Aim 1 we propose biochemical experiments and computational analyses to evaluate these possibilities. Furthermore, we will test the hypothesis that alternative splicing of overlapping 5' splice sites serves to reestablish reading the frame disrupted by additional splicing events. In Specific Aim 2, we will perform a similar set of computational and biochemical experiments to determine why 3' splice-site activation 3 nucleotides upstream or downstream of the dominant splice site is the most frequent form of alternative 3' splice-site usage within the human genome. [unreadable] [unreadable] [unreadable]